List of primers used in the paper

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Thalidomide and its derivatives are molecular glues that bind cereblon (CRBN), a component of an E3 ubiquitin ligase complex, and mediate protein interactions with neosubstrates resulting in their polyubiquitination and proteasomal degradation. The structural features of neosubstrate binding have been elucidated that highlight key interactions with a β-hairpin degron containing a glycine, which is present in a wide-range of proteins, including zinc-finger transcription factors such as IKZF1, and the translation termination factor GSPT1. Here, we profile 14 closely-related thalidomide derivatives in CRBN occupancy, and IKZF1 and GSPT1 degradation cell-based assays, and use crystal structures, computational docking and molecular dynamics to delineate subtle structure-activity relationships. Our findings will enable the rational design of CRBN modulators in the future, and help avoid the degradation of GSPT1 which is broadly cytotoxic.

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Targeted protein degradation (TPD) has emerged as a potentially transformational therapeutic modality with considerable promise. Molecular glue degraders remodel the surface of E3 ligases inducing interactions with neosubstrates resulting in their polyubiquitination and proteasomal degradation. Molecular glues are clinically precedented and have demonstrated the ability to degrade proteins‐of‐interest (POIs) previously deemed undruggable due to the absence of a traditional small molecule binding pocket. Heterobifunctional proteolysis targeting chimeras (PROTACs) possess ligands for an E3 complex and the POIs, which are chemically linked together, and similarly hijack the ubiquitin machinery to deplete the target. There has been a recent surge in the number of degraders entering clinical trials, particularly directed toward cancer. Nearly all utilize CRL4 CRBN as the E3 ligase, and a relatively limited diversity of POIs are currently targeted. In this review, we provide an overview of the degraders in clinical trials and provide a perspective on the lessons learned from their development and emerging human data that will be broadly useful to those working in the TPD field.

<p>Supplementary tables and figure legends</p>

miRNAs significantly changed after ENT treatment in H661 cells

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Electrophilic biocompatible warheads, particularly cysteine-reactive acrylamides, have enabled the development of covalent inhibitor drugs and chemical biology probes, but cysteine is rarely present in protein binding sites. Therefore, expansion of the list of targetable amino acid residues is required to augment the synthetic bology toolkit of site-selective protein modifications. This work describes the first rational targeting of a specific histidine residue in a protein binding site using sulfonyl exchange chemistry. Structure-based drug design was used to incorporate sulfonyl fluoride and triazole reactive groups into the isoindolinone thalidomide congener EM12 to yield potent covalent inhibitors of the cereblon E3 ubiquitin ligase complex through engagement of His353. Conversely, the fluorosulfate derivative EM12-FS labels His353, but degrades a novel neosubstrate, the protein N-terminal glutamine amidohydrolase NTAQ1, which is involved in the N-end rule pathway and DNA damage response. Targeted protein degradation using cereblon ligands has become an important new drug discovery modality and the chemical probes and covalent labeling strategy described here will broadly impact this exciting area of therapeutic research.

Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide engage cereblon and mediate a protein interface with neosubstrates such as zinc finger transcription factors promoting their polyubiquitination and degradation. The IMiDs have garnered considerable excitement in drug discovery, leading to exploration of targeted protein degradation strategies. Although the molecular modes-of-action of the IMiDs and related degraders have been the subject of intense research, their pharmacokinetics and disposition have been relatively understudied. Here, we assess the effects of physicochemistry of the IMiDs, the phthalimide EM-12, and the candidate drug CC-220 (iberdomide) on lipophilicity, solubility, metabolism, permeability, intracellular bioavailability, and cell-based potency. The insights yielded in this study will enable the rational property-based design and development of targeted protein degraders in the future.

The zinc finger transcription factor SALL4 is highly expressed in embryonic stem cells, downregulated in most adult tissues, but reactivated in many aggressive cancers. This unique expression pattern makes SALL4 an attractive therapeutic target. However, whether SALL4 binds DNA directly to regulate gene expression is unclear, and many of its targets in cancer cells remain elusive. Here, through an unbiased screen of protein binding microarray (PBM) and cleavage under targets and release using nuclease (CUT&RUN) experiments, we identify and validate the DNA binding domain of SALL4 and its consensus binding sequence. Combined with RNA sequencing (RNA-seq) analyses after SALL4 knockdown, we discover hundreds of new SALL4 target genes that it directly regulates in aggressive liver cancer cells, including genes encoding a family of histone 3 lysine 9-specific demethylases (KDMs). Taken together, these results elucidate the mechanism of SALL4 DNA binding and reveal pathways and molecules to target in SALL4-dependent tumors.

The architecture of chromatin regulates eukaryotic cell states by controlling transcription factor access to sites of gene regulation. Here we describe a dual transposase-peroxidase approach, integrative DNA and protein tagging (iDAPT), which detects both DNA (iDAPT-seq) and protein (iDAPT-MS) associated with accessible regions of chromatin. In addition to direct identification of bound transcription factors, iDAPT enables the inference of their gene regulatory networks, protein interactors and regulation of chromatin accessibility. We applied iDAPT to profile the epigenomic consequences of granulocytic differentiation of acute promyelocytic leukemia, yielding previously undescribed mechanistic insights. Our findings demonstrate the power of iDAPT as a platform for studying the dynamic epigenomic landscapes and their transcription factor components associated with biological phenomena and disease.

Abstract Oncofetal protein SALL4 is critical for cancer cell survival. Targeting SALL4, however, is only applicable in a fraction of cancer patients who are positive for this gene. To overcome this limitation, we propose to induce a cancer vulnerability by engineering a partial dependency upon SALL4. Following exogenous expression of SALL4, SALL4-negative cancer cells became partially dependent on SALL4. Treatment of SALL4-negative cells with the FDA-approved hypomethylating agent 5-aza-2′-deoxycytidine (DAC) resulted in transient upregulation of SALL4. DAC pretreatment sensitized SALL4-negative cancer cells to entinostat, which negatively affected SALL4 expression through a microRNA, miRNA-205, both in culture and in vivo. Moreover, SALL4 was essential for the efficiency of sequential treatment of DAC and entinostat. Overall, this proof-of-concept study provides a framework whereby the targeting pathways such as SALL4-centered therapy can be expanded, sensitizing cancer cells to treatment by transient target induction and engineering a dependency. Significance: These findings provide a therapeutic approach for patients harboring no suitable target by induction of a SALL4-mediated vulnerability.

Oncofetal protein SALL4 is critical for tumor cell survival, making it a promising target in cancer therapy. However, it is detectable only in a subset of cancer patients, which limits the therapeutic impact of a SALL4 targeted therapy. Here we report that SALL4 can be activated and/or upregulated pharmacologically by hypomethylating agents, such as 5-Aza-2’-deoxycytidine (DAC), which are used clinically, and that SALL4 negative cancer cells become SALL4 dependent following exogenous expression of SALL4. In addition, the histone deacetylase inhibitor Entinostat (ENT) negatively regulates SALL4 expression by upregulating miR-205. Both ENT and miR-205 treatment induced cell apoptosis, rescuable by SALL4 expression or miR-205 inhibition. Finally, DAC pre-treatment sensitizes SALL4 negative cancer cell lines to ENT both in culture and in vivo by upregulating SALL4. Overall, we propose a framework whereby the scope of targeted therapy can be expanded by sensitizing cancer cells to treatment by target induction and engineered dependency. Significance This proof of concept study demonstrates that targeted cancer therapy can be achieved by inducing a targetable gene establishing a survival-dependency for cancer cells. For SALL4, sequential treatment of DAC and ENT could expand the scope of SALL4 targeted cancer therapy.

Abstract Transcription factors are important drivers of cancer but the development of therapeutics against these factors has had limited success. We developed a stringent high-throughput chemical genetic screening platform to identify compounds that target oncogenic transcription factor SALL4 dependency in liver cancer. The platform comprises SALL4 low- and high-expressing endogenous and engineered isogenic liver cancer cell lines. Unexpectedly, from screening 21,575 natural product extracts, the top hits were four oxidative phosphorylation inhibitors that selectively reduced SALL4-dependent cell viability. The ATP synthase inhibitor oligomycin suppressed SALL4-expressing cancer in lung and liver cancer cell culture models, and in patient-derived xenograft models of liver cancer. Oligomycin also synergized with sorafenib, the standard-of-care targeted therapy in liver cancer, to effectively suppress SALL4-driven tumorigenesis in vivo. When aberrantly expressed in cancer, SALL4 binds ~50% of mitochondrial genes, including many oxidative phosphorylation genes, to predominantly upregulate their expression. SALL4 upregulation also alters the levels of oxidative phosphorylation-related metabolites and functionally increases oxidative phosphorylation activity. Application of our endogenous/isogenic transcription factor-screening platform revealed a therapeutically actionable oxidative phosphorylation vulnerability in SALL4-expressing cancers. Citation Format: Justin L. Tan, Feng Li, Joanna Z. Yeo, Kol Jia Yong, Mahmoud A. Bassal, Guo Hao Ng, May Yin Lee, Chung Yan Leong, Hong Kee Tan, Chan-Shuo Wu, Bee Hui Liu, Hon Man Chan, Zi Hui Tan, Yun Shen Chan, Siyu Wang, Zhi Han Lim, Tan Boon Toh, Lissa Hooi, Kia Ngee Low, Siming Ma, Nikki R. Kong, Alicia J. Stein, Yue Wu, Matan T. Thangavelu, Atsushi Suzuki, Giridharan Periyasamy, John M. Asara, Yock Young Dan, Glenn K. Bonney, Edward K. Chow, Guo-Dong Lu, Huck Hui Ng, Yoganathan Kanagasundaram, Siew Bee Ng, Wai Leong Tam, Li Chai, Daniel G. Tenen. A high-throughput chemical genetic screen reveals SALL4-induced metabolic vulnerabilities in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1788.

BACKGROUND & AIMS:Some oncogenes encode transcription factors, but few drugs have been successfully developed to block their activity specifically in cancer cells. The transcription factor SALL4 is aberrantly expressed in solid tumor and leukemia cells. We developed a screen to identify compounds that reduce the viability of liver cancer cells that express high levels of SALL4, and we investigated their mechanisms.METHODS:We developed a stringent high-throughput screening platform comprising unmodified SNU-387 and SNU-398 liver cancer cell lines and SNU-387 cell lines engineered to express low and high levels of SALL4. We screened 1597 pharmacologically active small molecules and 21,575 natural product extracts from plant, bacteria, and fungal sources for those that selectively reduce the viability of cells with high levels of SALL4 (SALL4hi cells). We compared gene expression patterns of SALL4hi cells vs SALL4-knockdown cells using RNA sequencing and real-time polymerase chain reaction analyses. Xenograft tumors were grown in NOD/SCID gamma mice from SALL4hi SNU-398 or HCC26.1 cells or from SALL4lo patient-derived xenograft (PDX) cells; mice were given injections of identified compounds or sorafenib, and the effects on tumor growth were measured.RESULTS:Our screening identified 1 small molecule (PI-103) and 4 natural compound analogues (oligomycin, efrapeptin, antimycin, and leucinostatin) that selectively reduced viability of SALL4hi cells. We performed validation studies, and 4 of these compounds were found to inhibit oxidative phosphorylation. The adenosine triphosphate (ATP) synthase inhibitor oligomycin reduced the viability of SALL4hi hepatocellular carcinoma and non-small-cell lung cancer cell lines with minimal effects on SALL4lo cells. Oligomycin also reduced the growth of xenograft tumors grown from SALL4hi SNU-398 or HCC26.1 cells to a greater extent than sorafenib, but oligomycin had little effect on tumors grown from SALL4lo PDX cells. Oligomycin was not toxic to mice. Analyses of chromatin immunoprecipitation sequencing data showed that SALL4 binds approximately 50% of mitochondrial genes, including many oxidative phosphorylation genes, to activate their transcription. In comparing SALL4hi and SALL4-knockdown cells, we found SALL4 to increase oxidative phosphorylation, oxygen consumption rate, mitochondrial membrane potential, and use of oxidative phosphorylation-related metabolites to generate ATP.CONCLUSIONS:In a screening for compounds that reduce the viability of cells that express high levels of the transcription factor SALL4, we identified inhibitors of oxidative phosphorylation, which slowed the growth of xenograft tumors from SALL4hi cells in mice. SALL4 activates the transcription of genes that regulate oxidative phosphorylation to increase oxygen consumption, mitochondrial membrane potential, and ATP generation in cancer cells. Inhibitors of oxidative phosphorylation might be used for the treatment of liver tumors with high levels of SALL4.

Hematopoietic stem cells are capable of self-renewal or differentiation along three main lineages: myeloid, erythroid, and lymphoid. One of the earliest lineage decisions for blood progenitor cells is whether to adopt the lymphoid or myeloid fate. Previous work had shown that myocyte enhancer factor 2C (MEF2C) is indispensable for the lymphoid fate decision, yet the specific mechanism of action remained unclear. Here, we have identified early B cell factor-1 (EBF1) as a co-regulator of gene expression with MEF2C. A genome-wide survey of MEF2C and EBF1 binding sites identified a subset of B cell-specific genes that they target. We also determined that the p38 MAPK pathway activates MEF2C to drive B cell differentiation. Mef2c knockout mice showed reduced B lymphoid-specific gene expression as well as increased myeloid gene expression, consistent with MEF2C's role as a lineage fate regulator. This is further supported by interaction between MEF2C and the histone deacetylase, HDAC7, revealing a likely mechanism to repress the myeloid transcription program. This study thus elucidates both activation and repression mechanisms, identifies regulatory partners, and downstream targets by which MEF2C regulates lymphoid-specific differentiation.

There is a growing body of evidence supporting that cancer cells share many similarities with embryonic stem cells (ESCs). For example, aggressive cancers and ESCs share a common gene expression signature that includes hundreds of genes. Since ESC genes are not present in most adult tissues, they could be ideal candidate targets for cancer-specific diagnosis and treatment. This is an exciting cancer-targeting model. The major hurdle to test this model is to identify the key factors/pathway(s) within ESCs that are responsible for the cancer phenotype. SALL4 is one of few genes that can establish this link. The first publication of SALL4 is on its mutation in a human inherited disorder with multiple developmental defects. Since then, over 300 papers have been published on various aspects of this gene in stem cells, development, and cancers. This review aims to summarize our current knowledge of SALL4, including a SALL4-based approach to classify and target cancers. Many questions about this important gene still remain unanswered, specifically, on how this gene regulates cell fates at a molecular level. Understanding SALL4's molecular functions will allow development of specific targeted approaches in the future.